A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9 and evaluation of antisense-mediated exon skipping

AbstractDuchenne muscular dystrophy (DMD) is an X-linked progressive muscle-wasting disorder that is caused by a lack of functional dystrophin, a cytoplasmic protein necessary for the structural integrity of muscle. As variants in the dystrophin gene lead to a disruption of the reading frame, pharmacological treatments have only limited efficacy; there is currently no effective therapy and consequently, a significant unmet clinical need for DMD. Recently, novel genetic approaches have shown real promise in treating DMD, with advancements in the efficacy and tropism of exon skipping and surrogate gene therapy. CRISPR-Cas9 has the potential to be a ‘one-hit’ curative treatment in the coming decade. The current limitations of gene editing, such as off-target effects and immunogenicity, are in fact partly constraints of the delivery method itself, and thus research focus has shifted to improving the viral vector. In order to halt the loss of ambulation, early diagnosis and treatment will be pivotal. In an era where genetic sequencing is increasingly utilised in the clinic, genetic therapies will play a progressively central role in DMD therapy. This review delineates the relative merits of cutting-edge genetic approaches, as well as the challenges that still need to be overcome before they become clinically viable.

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Restoring Protein Expression in Neuromuscular Conditions: A Review Assessing the Current State of Exon Skipping/Inclusion and Gene Therapies for Duchenne Muscular Dystrophy and Spinal Muscular Atrophy

BioDrugs ◽

10.1007/s40259-021-00486-7 ◽

2021 ◽

Author(s):

Omar Sheikh ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Protein Expression ◽

Spinal Muscular Atrophy ◽

Muscular Atrophy ◽

Exon Skipping ◽

Gene Therapies ◽

Current State ◽

Neuromuscular Conditions

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The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01106-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Dominic Scaglioni ◽

Francesco Catapano ◽

Matthew Ellis ◽

Silvia Torelli ◽

Darren Chambers ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotide ◽

De Novo ◽

Exon Skipping ◽

Significant Negative Correlation ◽

Entire Cohort ◽

Associated Proteins ◽

Analysis Platform ◽

Dystrophin Protein

AbstractDuring the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

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Open-Label Evaluation of Eteplirsen in Patients with Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping: PROMOVI Trial

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210643 ◽

2021 ◽

pp. 1-13

Author(s):

Craig M. McDonald ◽

Perry B. Shieh ◽

Hoda Z. Abdel-Hamid ◽

Anne M. Connolly ◽

Emma Ciafaloni ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Natural History ◽

Adverse Events ◽

Exon Skipping ◽

Control Group ◽

Phase 2 ◽

Open Label ◽

M Phase ◽

Positive Treatment

Background Eteplirsen received accelerated FDA approval for treatment of Duchenne muscular dystrophy (DMD) with mutations amenable to exon 51 skipping, based on demonstrated dystrophin production. Objective To report results from PROMOVI, a phase 3, multicenter, open-label study evaluating efficacy and safety of eteplirsen in a larger cohort. Methods Ambulatory patients aged 7–16 years, with confirmed mutations amenable to exon 51 skipping, received eteplirsen 30 mg/kg/week intravenously for 96 weeks. An untreated cohort with DMD not amenable to exon 51 skipping was also enrolled. Results 78/79 eteplirsen-treated patients completed 96 weeks of treatment. 15/30 untreated patients completed the study; this cohort was considered an inappropriate control group because of genotype-driven differences in clinical trajectory. At Week 96, eteplirsen-treated patients showed increased exon skipping (18.7-fold) and dystrophin protein (7-fold) versus baseline. Post-hoc comparisons with patients from eteplirsen phase 2 studies (4658-201/202) and mutation-matched external natural history controls confirmed previous results, suggesting clinically notable attenuation of decline on the 6-minute walk test over 96 weeks (PROMOVI: –68.9 m; phase 2 studies: –67.3 m; external controls: –133.8 m) and significant attenuation of percent predicted forced vital capacity annual decline (PROMOVI: –3.3%, phase 2 studies: –2.2%, external controls: –6.0%; p < 0.001). Adverse events were generally mild to moderate and unrelated to eteplirsen. Most frequent treatment-related adverse events were headache and vomiting; none led to treatment discontinuation. Conclusions This large, multicenter study contributes to the growing body of evidence for eteplirsen, confirming a positive treatment effect, favorable safety profile, and slowing of disease progression versus natural history.

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In vivocomparison of 2′-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

The Journal of Gene Medicine ◽

10.1002/jgm.1288 ◽

2009 ◽

Vol 11 (3) ◽

pp. 257-266 ◽

Cited By ~ 128

Author(s):

Hans A. Heemskerk ◽

Christa L. de Winter ◽

Sjef J. de Kimpe ◽

Petra van Kuik-Romeijn ◽

Niki Heuvelmans ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotides ◽

Exon Skipping

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The Interplay of Mitophagy and Inflammation in Duchenne Muscular Dystrophy

Life ◽

10.3390/life11070648 ◽

2021 ◽

Vol 11 (7) ◽

pp. 648

Author(s):

Andrea L. Reid ◽

Matthew S. Alexander

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mitochondrial Dysfunction ◽

Disease Onset ◽

Exon Skipping ◽

Dystrophin Gene ◽

Muscle Disease ◽

Mitochondrial Morphology ◽

Gene Therapies ◽

Dystrophin Protein

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

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